ABSTRACTCapsazepine (CPZ) inhibits Na+,K+-ATPase-mediated K+-dependent ATP hydrolysis with no effect on Na+-ATPase activity. In this study we have investigated the functional effects of CPZ on Na+,K+-ATPase in intact cells. We have also used well established biochemical and biophysical techniques to understand how CPZ modifies the catalytic subunit of Na+,K+-ATPase. In isolated rat cardiomyocytes, CPZ abolished Na+,K+-ATPase current in the presence of extracellular K+. In contrast, CPZ stimulated pump current in the absence of extracellular K+. Similar conclusions were attained using HEK293 cells loaded with the Na+ sensitive dye Asante NaTRIUM green. Proteolytic cleavage of pig kidney Na+,K+-ATPase indicated that CPZ stabilizes ion interaction with the K+ sites. The distal part of membrane span 10 (M10) of the α-subunit was exposed to trypsin cleavage in the presence of guanidinum ions, which function as Na+ congener at the Na+ specific site. This effect of guanidinium was amplified by treatment with CPZ. Fluorescence of the membrane potential sensitive dye, oxonol VI, was measured following addition of substrates to reconstituted inside-out Na+,K+-ATPase. CPZ increased oxonol VI fluorescence in the absence of K+, reflecting increased Na+ efflux through the pump. Surprisingly, CPZ induced an ATP-independent increase in fluorescence in the presence of high extravesicular K+, likely indicating opening of an intracellular pathway selective for K+. As revealed by the recent crystal structure of the E1.AlF4-.ADP.3Na+ form of the pig kidney Na+,K+-ATPase, movements of M5 of the α-subunit, which regulate ion selectivity, are controlled by the C-terminal tail that extends from M10. We propose that movements of M10 and its cytoplasmic extension is affected by CPZ, thereby regulating ion selectivity and transport through the K+ sites in Na+,K+-ATPase.

pone-0096909-g004: Effect of temperature, pH, and vanadate sensitivity of the CPZ treated enzyme.A. Pig kidney Na+,K+-ATPase activity was measured at four different temperatures, as described in Materials and Methods, in the presence of the indicated CPZ concentrations. The reaction contained 30 mM histidine buffer, pH 7.2, 100 mM NaCl, 20 mM KCl, 3 mM MgCl2, and 3 mM ATP. The inhibition curves were analyzed using the Hill equation, giving the following IC50 values; squares, 5°C (7.64±1.14 µM); circles, 15°C (13.88±1.05 µM); diamonds, 30°C (37.33±1.08 µM); and triangles, 37 °C (108.20±1.31 µM). B. Effect of pH on CPZ inhibition of Na+-ATPase activity. Na+-ATPase activity was measured as described in panel A, but in the absence of K+ and in the presence of different pH values (the reaction was buffered with 30 mM Tris adjusted with HCl), in the presence of the indicated CPZ concentrations. Squares, circles, and diamonds indicate measurements performed at pH 6, 7, or 8, respectively. C. Na+,K+-ATPase (NKA) activity was measured as in panel A, but in the presence of 1 mM ATP and the indicated vanadate concentrations at 23°C. The ATPase mixture contained either DMSO (squares) or 20 µM CPZ (circles).

pone-0096909-g004: Effect of temperature, pH, and vanadate sensitivity of the CPZ treated enzyme.A. Pig kidney Na+,K+-ATPase activity was measured at four different temperatures, as described in Materials and Methods, in the presence of the indicated CPZ concentrations. The reaction contained 30 mM histidine buffer, pH 7.2, 100 mM NaCl, 20 mM KCl, 3 mM MgCl2, and 3 mM ATP. The inhibition curves were analyzed using the Hill equation, giving the following IC50 values; squares, 5°C (7.64±1.14 µM); circles, 15°C (13.88±1.05 µM); diamonds, 30°C (37.33±1.08 µM); and triangles, 37 °C (108.20±1.31 µM). B. Effect of pH on CPZ inhibition of Na+-ATPase activity. Na+-ATPase activity was measured as described in panel A, but in the absence of K+ and in the presence of different pH values (the reaction was buffered with 30 mM Tris adjusted with HCl), in the presence of the indicated CPZ concentrations. Squares, circles, and diamonds indicate measurements performed at pH 6, 7, or 8, respectively. C. Na+,K+-ATPase (NKA) activity was measured as in panel A, but in the presence of 1 mM ATP and the indicated vanadate concentrations at 23°C. The ATPase mixture contained either DMSO (squares) or 20 µM CPZ (circles).

ABSTRACTCapsazepine (CPZ) inhibits Na+,K+-ATPase-mediated K+-dependent ATP hydrolysis with no effect on Na+-ATPase activity. In this study we have investigated the functional effects of CPZ on Na+,K+-ATPase in intact cells. We have also used well established biochemical and biophysical techniques to understand how CPZ modifies the catalytic subunit of Na+,K+-ATPase. In isolated rat cardiomyocytes, CPZ abolished Na+,K+-ATPase current in the presence of extracellular K+. In contrast, CPZ stimulated pump current in the absence of extracellular K+. Similar conclusions were attained using HEK293 cells loaded with the Na+ sensitive dye Asante NaTRIUM green. Proteolytic cleavage of pig kidney Na+,K+-ATPase indicated that CPZ stabilizes ion interaction with the K+ sites. The distal part of membrane span 10 (M10) of the α-subunit was exposed to trypsin cleavage in the presence of guanidinum ions, which function as Na+ congener at the Na+ specific site. This effect of guanidinium was amplified by treatment with CPZ. Fluorescence of the membrane potential sensitive dye, oxonol VI, was measured following addition of substrates to reconstituted inside-out Na+,K+-ATPase. CPZ increased oxonol VI fluorescence in the absence of K+, reflecting increased Na+ efflux through the pump. Surprisingly, CPZ induced an ATP-independent increase in fluorescence in the presence of high extravesicular K+, likely indicating opening of an intracellular pathway selective for K+. As revealed by the recent crystal structure of the E1.AlF4-.ADP.3Na+ form of the pig kidney Na+,K+-ATPase, movements of M5 of the α-subunit, which regulate ion selectivity, are controlled by the C-terminal tail that extends from M10. We propose that movements of M10 and its cytoplasmic extension is affected by CPZ, thereby regulating ion selectivity and transport through the K+ sites in Na+,K+-ATPase.